The Origin Recognition Complex (ORC) has two known DNA binding functions in Saccharomyces cerevisiae, DNA replication initiation and silent chromatin assembly. The focus of this proposal is to determine the mechanism that distinguishes these two functions. Hypothesis: Specific ORC-DNA interactions are optimized for silent chromatin assembly Aim 1: Determine the DNA sequences that distinguish silencing from replication initiation. In vitro binding assays will be used to determine the affinity of known ORC replication origins and silencer elements. High affinity ORC binding sites will be identified using SELEX and then assayed for silencing and origin activity. Aim 2: Identify and examine new alleles of ORC genes defective in silencing but competent for genome replication. Mutants will be isolated in targeted genetic screens and analyzed in vitro for DNA binding and recruitment of the essential silencing factor, Sir1 p. Together, these experiments lay foundation for advancing our understanding of origin activity and regulation in all cells, including both healthy dividing cells such as stem cells and aberrantly proliferating cells associated with cancers.